Screening system

ABSTRACT

The present invention concerns a screening system for screening effector peptides that can act as agonists or antagonists, as well as means to produce such a screening system, in particular a specific DNA library encoding peptides. In order to perform the screening, host cells comprising a selection system are transformed with the DNA library and the thus obtained screening system is then cultivated under conditions selectively allowing survival of cells with desired interaction.

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims the priority of European patent application no.98111428.3, filed Jun. 22, 1998, the disclosure of which is incorporatedherein by reference in its entirety.

TECHNICAL FIELD

The present invention concerns a screening system, to identify leadcompounds. Said screening system is especially suitable for thescreening of small molecules, whereby peptides are the much preferredsmall molecules.

BACKGROUND ART

Screening compounds in order to find molecules for further drugdevelopment, is one of the crucial steps in such drug development. Up tonow, product screening is performed in vitro, e.g. by so calledhigh-throughput screening systems or by phage display. Phage displaysystems can identify proteins or peptides which are expressed on thesurface of a bacteriophage particle (see R. Cortese et al., Selection ofbiologically active peptides by phage display of random peptidelibraries, Current Opinion in Biotechnology 1996, 7, 616-621, 1996).Only in the case of a successful interaction between the two proteins,the bacteriophage can infect the bacterial E. coli host. This eventbecomes visible since this successful bacteriophage will spread and formplaques which consist of lysed E. coli cells. High throughput screeningsystems suitable for the screening of small molecules are e.g. describedin WO 9710253 by Merck & Co. Inc. Such systems are generally applied forthe screening of e.g. plant extracts, etc. in vitro.

These screening systems of the state of the art are very laborious andtime-consuming.

The disadvantage of these screening systems of the state of the art isthat they are far away from natural conditions and very slow. Inparticular, no post-translational modifications of proteins occur in thephage display system. Additionally, the interactions of the phage systemoccur in vitro, whereas the interesting protein—protein interactions inmammals, including man, actually occur in the cell, many of them in thenucleus.

It is also known to study protein—protein interactions in vivo by usingtwo hybrid proteins. The protein of interest is fused to a DNA bindingdomain, e.g. a GAL4 DNA binding domain, whereas a library of potentialinteraction candidates is fused to a transcription activation domain,e.g. a GAL4 transcription activation domain. Only in the case ofsuccessful interaction between the protein of interest and a realinteracting candidate, the transcriptional activation domain is broughtto the reporter gene (lacZ), which leads to a blue colouring of thiscell (see Fields and Song, A novel genetic system to detectprotein—protein interactions, Nature 340, 245, 1989). Such systems areadditionally described in reviews and patent literature (see e.g. S.Fields and R. Sternglanz, The Two-hybrid System: An Essay forProtein-protein Interactions, Reviews, TIG August 1994, Vol. 10, No. 8,p. 286-292, and The Matchmaker Two-hybrid System, published by Clontech,as well as U.S. Pat. Nos. 5,283,173 and 5,468,614 both assigned to theResearch Foundation of the State University of New York). A furthertwo-hybrid system, which is a modification of the classical one, andwhich—due to the modification—is dependent on post-translationalmodifications of one of the proteins, is described in U.S. Pat. No.5,637,463. Such two-hybrid systems are disclosed to have three majorapplications: Testing known proteins for interaction, defining domainsor amino acids critical for an interaction, and screening libraries forproteins that bind some target protein. It is also known to modify theexisting classical two-hybrid screening. Instead of fusing a library ofcellular proteins to an activation domain, peptide libraries are fusedto the GAL4 activation domain (see WO97/41255, WO95/34646, WO92/05286,FR 2720068). It is also known to fuse a peptide library to a DNA bindingdomain (see U.S. Pat. No. 5,498,530). The selection principle isidentical to the classical two-hybrid (see Young, Wo and Fields,Protein—peptide interactions analysed with the yeast two-hybrid system,Nucl. Acids Res. 23, 1152, 1995).

Another DNA-binding domain and activation domains e.g. disclosed inconnection with screening for peptides which interact with the cyclindependent kinase II. Here, the Cdk2 was fused to a LexA DNA bindingdomain, and the peptide library was cloned into the active loop of theE. coli thioredoxin, which was also fused to an activation domain (seeColas, Cohen, Jessen, Grishina, McCoy & Brent, Genetic selection ofpeptide aptamers that recognise and inhibit cyclin-dependent kinase II,Nature, 30, 554, 1996 and WO 94/10300). Information about other reportergenes can be found in Vidal, Brachmann, Fatai, Harlow and Böcke,Reversed two-hybrid to detect dissociation of protein—protein and DNAprotein interactions, Proc. Natl. Acad. Sci. USA 93, 10315, 1996, andVidal, Brown, Chen, Böcke and Harlow, Genetic characterisation of amammalian protein—protein interaction domain by using a yeast reversetwo-hybrid system, Proc. Natl. Acad. Sci. USA 93, 10321, 1996.

Other two-hybrid systems were described by A. Aronheim et al., Isolationof an AP-1repressor by a novel method for detecting protein—proteininteractions, Mol. Cell. Biol. 17, 3094-3102, 1997; and by I. Stagljaret al., A genetic system based on split-ubiquitin for the analysis ofinteractions between membrane proteins in vivo, Proc. Natl. Acad. Sci.USA 95, 5187-5192, 1998. In the Aronheim report a two-hybrid systembased on the mammalian GDP-GTP exchange factor (GEF)hSoS is described.

Also known are so-called three-hybrid systems to detect RNA-proteininteractions (see e.g. D. J. SenGupta et al., A three-hybrid system todetect RNA-protein interactions in vivo, Proc. Acad. Natl. Sci. USA 93,8496-8501, 1996), or mediating proteins (WO97/24457).

The two-hybrid method, as it is described in the above mentionedliterature, thus allows to study molecular interactions that might bethe causes of diseases. However, it does not show how to use suchsystems in the search for active substances and the development of noveldrugs.

It is also already known to use the two-hybrid technology to findexogenous small molecules, which inhibit protein—protein interaction(see WO97/41255, U.S. Pat. No. 5,569,588, WO92/05286). Hybrid proteins,of which the interaction should be disrupted, can also be expressed asfusions to a DNA binding domain (LexA) and an activation domain (B42).The small molecule can be covalently linked to a polymer bead, which isphotoreleasable from the small molecule (see Huang & Schreiber, A yeastgenetic system for selecting small molecule inhibitors ofprotein—protein interaction in nano droplets, Proc. Natl. Acad. Sci. USA94, 1336, 1997).

This technology is not suitable for a screening of interacting peptides,since it is laborious in view of the production of peptides, and theanalysis of the possibly interacting peptides.

Attempts to screen HIV-1 Rev protein inhibitors have already been made(U.S. Pat. No. 5,691,137 to Brandeis University, Waltham, Mass.). Insaid attempts the re-porter gene used was CUP 1 modified by insertion ofan intron sequence, and a HIV-1 Rev response element 3′ of the CUP 1open reading frame. Upon interaction of Rev with its response element,the splicing of the pre-mRNA is inhibited leading to a inactive protein.

DISCLOSURE OF THE INVENTION

Hence, it is a general object of the invention to provide a much fasterscreening system allowing the trial of many different effector peptideswith agonist or antagonist effect in parallel, whereby the time andcosts of the first step in drug development can be dramatically reduced.

This object is achieved by providing a screening system for agonists orantagonists comprising a eukaryotic host cell stably transformed with aselection system enabling the survival of the cells in the case ofdesired interaction between at least one target protein and an effectorpeptide, and a peptide expressing system,

whereby said selection system comprises

at least one monitoring gene enabling the detection of said host cellupon transcription of said monitoring gene, said at least one monitoringgene being directly or indirectly under the control of a specificactivation system and,

at least one DNA sequence coding for at least one target molecule, saidone or more target molecule(s) being selected from the group comprisingRNA sequences or proteins, said one or more target molecule(s) beingresponsible in their natural environment for the induction of theproduction and/or activity of an undesired protein or the omission ofthe production and/or activation of a desired protein, said at least oneDNA sequence coding for said at least one target molecule being underthe control of an host cell specific active promoter, preferably a hostcell specific promoter,

whereby said specific activation system is selectively modulated (i.e.activated or inactivated) in the presence of specific interaction(s)between at least one of said target protein(s) and an effector peptide,

whereby said specific activation system upon modulation directly orindirectly modulates the transcription of at least one monitoring geneenabling the survival of said host cell, and

whereby said effector peptide expressing system comprises a peptideencoding nucleic acid sequence under the control of an active promoter,preferably a host cell specific active promoter.

In a preferred embodiment, said at least one monitoring gene is anucleic acid sequence encoding at least one monitoring protein enablingthe detection of said host cell upon expression of said at least onemonitoring protein, said at least one nucleic acid encoding said atleast one monitoring protein being under the control of said specificactivation system, and whereby said specific activation system uponmodulation modulates the expression of at least one monitoring proteinenabling the survival of said host cell,

Other objects of the present invention are a screening method, a methodfor the production of a screening system, a DNA library as peptidesource, and a method to produce such library.

BRIEF DESCRIPTION OF THE DRAWINGS

The Invention will be better understood and objects other than those setforth above will become apparent when consideration is given to thefollowing detailed description thereof. Such description makes referenceto the annexed drawings, wherein:

FIG. 1 is a representation of the generation of a DNA library suitablefor peptide expression in yeast.

FIG. 2 shows the action of p53.

FIG. 3 shows the effect of mutant p53.

FIG. 4 shows the essential features of a screening system of the presentinvention for the screening of a mutant p53 agonist.

FIG. 5 represents the action of vascular endothelial growth factor inthe presence of a tumor on the level of induction of angiogenesis.

FIG. 6 represents a later state of the system represented in FIG. 5,namely tumor growth and metastasis formation due to angiogenesis.

FIG. 7 represents the situation during the screening of a host cell inthe absence of an antagonist.

FIG. 8 represents the situation during the screening of a host cell inthe presence of an antagonist.

FIG. 9 shows expression vector pESBA-ADH.

FIG. 10 shows expression vector pESBA-ACT.

FIG. 11 shows expression vector pESBA-GAL.

FIG. 12 shows reporter vector pESBA-HIS.

FIG. 13 shows reporter vector pESBA-URA.

FIG. 14 shows reporter vector pESBA-URA-17mer.

MODES FOR CARRYING OUT THE INVENTION

As mentioned above, a general object of the present invention is toprovide a much faster screening system for agonists or antagonistsallowing the trial of many different effector peptides in parallel,whereby the time and costs of the first step in drug development can bedramatically reduced. This object is achieved by providing a screeningsystem for agonists or antagonists comprising a eukaryotic host cellstably transformed with a selection system enabling the survival of thecells in the case of desired interaction between at least one targetprotein and an effector peptide, and a peptide expressing system,

whereby said selection system comprises

at least one monitoring gene enabling the detection of said host cellupon transcription of said monitoring gene, said at least one monitoringgene being directly or indirectly under the control of a specificactivation system and,

at least one DNA sequence coding for at least one target molecule, saidone or more target molecule(s) being selected from the group comprisingRNA sequences or proteins, said one or more target molecule(s) beingresponsible in their natural environment for the induction of theproduction and/or the activity of an undesired protein or the omissionof the production and/or the activation of a desired protein, said atleast one DNA sequence coding for said at least one target moleculebeing under the control of an host cell specific active promoter,preferably a host cell specific promoter,

whereby said specific activation system is selectively modulated (i.e.activated or inactivated) in the presence of specific interaction(s)between at least one of said target protein(s) and an effector peptide,

whereby said specific activation system upon modulation directly orindirectly modulates the transcription of at least one monitoring geneenabling the survival of said host cell, and

whereby said effector peptide expressing system comprises a peptideencoding nucleic acid sequence under the control of an active promoter,preferably a host cell specific active promoter.

In a preferred embodiment, said at least one monitoring gene is anucleic acid sequence encoding at least one monitoring protein enablingthe detection of said host cell upon expression of said at least onemonitoring protein, said at least one nucleic acid encoding said atleast one monitoring protein being under the control of said specificactivation system, and whereby said specific activation system uponmodulation modulates the expression of at least one monitoring proteinenabling the survival of said host cell.

Monitoring genes suitable for the present invention are e.g. genesencoding defined proteins the activity of which can be directlymonitored.

The modulation of the transcription means that transcription can eitherbe activated or inactivated, and direct modulation, refer so the directeffect on the transcription of the monitoring gene whereas indirectmodulation refers to a stimulation of cellular signals resulting in cellgrowth due to activation of cell-cycle regulatory genes.

Inventive screening systems allow the screening of effector peptideswith agonist as well as antagonist activity.

In order to prepare a screening system according to the presentinvention, information about relevant interactions, influenced by theeffector peptide, and preferably the causes, or at least a part of saidcauses, of the respective disease to be treated by the effector peptideitself, or an active substance derived from or developed on the bases ofsuch effector peptide have to be known.

On the basis of such knowledge, a selection principle according to thepresent invention, applicable according to the method of the presentinvention, is produced.

Different causes for diseases exist, such as

expression of a defective protein no longer able to activate theexpression of another essential protein,

expression of proteins interacting with each other or with nucleicacid(s), whereby said interaction(s) lead(s) to an undesired response inthe cell,

expression of a defective protein such, that a necessary protein—proteinor protein-nucleic acid interaction is impossible,

expression of a protein activating an undesired cell response.

The screening system of the present invention allows to find peptidesinteracting with at least one of such proteins or nucleic acids in amanner enabling a “normal” cell function.

The screening system of the present invention is suitable for theidentification of agonists and/or antagonists modulating molecularinteractions and molecular conformation, such as e.g. protein—proteininteractions, protein-RNA interactions, protein-DNA interactions. Inparticular, agonists and antagonists to protein—protein and protein-DNAinteractions are easily studied. The screening system of course is alsosuitable for optimizing specific peptides, e.g. already known peptides,in that a library encoding modifications is used. Such a library can beproduced by mutating the peptide encoding DNA sequence.

A preferred eukaryotic host cell is a yeast cell. Yeast cells are knownto perform similar co-translational and post-translational modificationsas mammalian cells. Additionally, yeast cells have the advantage of theease of transformation, the convenience of retrieving plasmids and theavailability of nutritional markers and well-characterised monitoringprotein expressing selector/reporter genes for direct selection.Furthermore, endogenous yeast proteins are less likely to bind amammalian protein, the binding of which would reduce the reliability ofthe screening system. However, the system of the present invention isnot limited to yeast cells. In particular, where specifically folded,glycosilated or otherwise modified proteins or peptides might be atissue, other mammalian cells can be used.

While also other small molecules can be screened by host cellscomprising at least one nucleic acid sequence encoding at least onemonitoring protein and at least one nucleic acid sequence encoding atleast one target protein as described above, the screening system of thepresent invention is in particular suitable in cases where theinteracting molecules, i.e. the effector molecules, are peptides. Inthis case it has been found that a very efficient screening is obtained,if peptide encoding DNA sequences are introduced into the cells. Suchintroduction is best performed by generating a DNA library. Such DNAlibrary can e.g. be obtained by fragmentation of genomic DNA or cDNA bydigestion or, preferably, by sonication and digestion with severalrestriction enzymes, or by synthesis of random oligo-nucleotides or, inparticular in the case where known peptide agonists or antagonists shallbe improved, by mutation of respective peptide encoding DNA. Therespectively obtained DNA fragments are then introduced into plasmidvectors, so that they are under the control of a host cell specificactive promoter (see FIG. 1). Further to the expression of the peptidesas such, they can be expressed fused to defined proteins. Such definedproteins preferably have no own activity in the screening system. Suchmethods of processing DNA fragments are known from general DNA librarytechnology. Such a DNA library with sequences encoding peptides (peptidelibrary) is also part of the present invention. Preferred peptidelibraries are those, wherein each peptide encoding sequence isincorporated in a separate plasmid vector, said plasmid vector enablingtransformation of the respective host cell and episomal expression ofthe peptide.

The host cells, comprising a selection system are then brought intocontact with such an inventive DNA library, preferably in a manner thatone peptide encoding sequence, e.g. one peptide encoding sequencecomprising plasmid, is introduced per cell.

In the case of host cell survival, showing the presence of a desiredinteraction, the peptide encoding DNA comprised in the surviving cellscan be isolated, analysed and multiplied to get information on theinteracting peptide. Additionally, such surviving yeast cells, or thecloned DNA in other production cells, can be used to produce theinteracting peptide, i.e. the effector peptide.

The advantage of the inventive screening method thus is, that billionsof different small peptides can easily be generated and analysed, andthat the method additionally provides a system allowing inexhaustibleproduction of the peptide of interest, or all means necessary to preparesuch a system, respectively.

Another important part of the present invention is the selection system,with which the host cells are transformed. Such selection systems arenow further described for each of the above mentioned types of defectsor in view of the desired molecule to be found, respectively.

Searching for agonists to one defective protein

Many diseases are due to the fact that an essential protein is notexpressed because of a defect in a transcription activating protein. Thescreening system of the present invention is suitable to search forsmall molecules able to “restore” the activating abilities of suchdefective proteins. For such screenings, the monitoring system comprisesa transcription activating sequence that is activated upon contact witha “restored” protein and is responsible for the expression of a proteinessential for the survival of the cell under the respective selectionconditions.

A host cell for this specific application is produced in that it istransformed with a DNA sequence, encoding a defective protein to berestored and additionally with a selector/reporter gene enabling thesurvival of the cell in a specific selection medium if expressed, saidselector/reporter gene being under the control of a transcriptionactivating sequence activated by the “restored” protein. Such a stablytransformed host cell can then be brought into contact with a DNAlibrary encoding a plurality of peptides.

One of such transcription activating proteins is the p53 tetramer. p53is a tumor suppressor gene which encodes a sequence-specific DNA-bindingtranscription factor. Through its ability to bind DNA, the p53 proteinis responsible for the activation of specific genes required for cellcycle arrest or apoptosis, i.e. the “suicide” of a cell which has beendamaged by e.g. cancerogenic substances (see FIG. 2). In many tumorcells, p53 has been mutated such that it no longer binds DNA, and it istherefore unable to activate the apoptotic genes. The loss of p53activity and the consequent lack of apoptosis leads to uncontrolledgrowth of cancer cells and therefore to tumor development (see FIG. 3).Mutations in p53 that abolish DNA binding are thought to change theshape of this protein so that it can no longer perform its naturalfunction. It has been shown that molecules such as antibodies andsynthetic peptides that can touch mutated p53 at specific sites, canreshape this protein so that it regains its natural conformation andtherefore its DNA binding function (see Selivanova et al. Restaurationof the Growth Suppression Function . . . , Nature Medicine, Vol. 3 No.6, 632 ff., 1997).

In such cases it is therefore very desirable to have a method to screena lot of peptides in order to find suitably interacting peptides.

In the case of the mutant p53 protein the inventive selection systemworks as follows;

p53 can be expressed in yeast cells where it can activate genes in asequence-specific manner. Similarly to what happens in human cells, themutated forms of p53 that are no longer capable of binding DNA cannotactivate genes in yeast. This property of yeast cells can be exploitedin the inventive screening system. The natural, non-mutated form of p53can activate the expression of an artificial gene in yeast which isessential for cell growth. As a consequence of this natural p53activity, and only in this case, yeast colonies will appear on anappropriate terrain. On the contrary, the mutant form of p53 which haslost the ability to bind DNA, cannot activate the expression of theartificial gene in yeast. As a consequence of this lack of activity,yeast cells containing the mutant p53 will not be able to grow and toform colonies on the appropriate terrain. In these cells, a smallpeptide with the ability to interact with the mutant p53, and to restoreits natural activity will cause activation of the artificial gene by themutant p53 (see FIG. 4). Expression of the artificial, essential genewill allow growth of the yeast cells and, therefore, formation ofcolonies.

In order to easily get information on the respective peptide being ableto restore the p53 action, the suitable host cells are brought intocontact with a peptide library. Briefly put, the respective procedure isas follows. Random, short DNA sequences, which are generated byfragmentation of natural DNA molecules or by synthesis are spliced intoso-called yeast expression vectors (see FIG. 1). This procedure cangenerate billions of different DNA sequences (DNA fragments) carried bybillions of expression vectors (see FIG. 1). These DNA vectors are thenintroduced into billions of yeast cells, such that in general each cellreceives one individual DNA fragment. Inside yeast cells, the differentrandom DNA sequences (DNA fragments) are expressed as part of genes toproduce an equivalent high number of random peptides. Due to thespecific monitoring systems, only those yeast cells that producepeptides capable of reactivating the mutant p53, which in turn activatesthe artificial gene, can grow to form colonies.

Growing colonies are then further cultivated, and the expression vectormolecule carrying the DNA sequence that generated the active peptide canbe extracted from the yeast cells for characterisation. DNA moleculescan be quickly characterised by sequence analysis, and the amino acidsequence of the active peptide can be easily deduced. Because theseisolated DNA molecules are clones, they can be multiplied indefinitelyand they directly represent an inexhaustible source of the activepeptides, the effector peptides.

As it is shown above with reference to p53, the screening system of thepresent invention is easily applicable in such cases where the bindingsite and the defective protein are known. Knowing the defective proteinof course allows to use a sequence encoding such defective protein whichis adapted to the specific host cell.

Screening for antagonists to suppress protein—protein interaction

It is also known that some responses in cells are activated byprotein—protein interactions. If natural regulation of such interactionis lost or modified, this could, for example, result in undue cellularproliferation that might lead to tumor formation, or faster tumorgrowth, or metastasis formation. It is thus desirable to findantagonists suitable to block such interactions. Such antagonists canalso be small peptides, advantageously detectable with the inventivescreening system.

The host cells suitable for such antagonist search are produced bytransforming the host cell with sequences for at least two, andpreferably two, hybrids, one such sequence encoding a first proteinconnected to a DNA-binding unit (in the case of polymerase II (pol II)activation a DNA-binding domain) under the control of an activepromoter, and a second sequence encoding a second protein linked to anactivation unit (in the case of pol II activation an activation domain),said sequence also being under the control of an active promoter. Theprocedure is now further described in terms of pol II activation. TheDNA-binding domain is suitable to bind to a transcription activatingsystem activating a selector/reporter gene. The transcription, however,is only activated if the binding domain and the activation domaininteract. Such interaction, however, is only obtained if the twoproteins connected to such domains are also interacting (see FIG. 7).The reporter gene suitable for such selection is a gene causing thedeath of the yeast cells upon activation, so that only such cells areable to survive, where the protein—protein interaction and thus thetranscription of the protein causing the death of yeast cells is blocked(see FIG. 8).

The accordingly transformed yeast cells are then brought into contactwith a DNA library as disclosed above, and those cells, producing ablocking peptide, are selected and analysed as described in more detailwith regard to the p53 system.

Such a system is e.g. very suitable for the antagonist search in thecase of the overexpression of specific proteins, such as e.g. theVascular Endothelial Growth Factor (VEGF). VEGF is a secreted proteinthat, by interacting with its receptors present on the surface ofendothelial cells forming the blood vessels, stimulates theirproliferation, the formation of new blood vessels. VEGF plays a pivotalrole in the growth of tumors because such growth requires the formationof new blood vessels to gain the necessary nutrients (see FIGS. 5 and6). Thus, blocking the function of VEGF in tumors would stop bloodvessel proliferation, which would lead to tumor shrinking and reducedmalignancy.

In yeast cells, the VEGF-receptor interaction is reproduced in a waythat it leads to the activation of an artificial gene. In contrast tothe selectable positive gene used in the p53 case, activation of thisartificial negative gene leads to death of the yeast cell in thepresence of a chemical compound. In other words, interaction between twomolecules, such as VEGF and its receptor, and the consequent artificialgene activation, produces no colonies on the appropriate terraincontaining the chemical compound. In these yeast cells, as described forp53, expression vectors carrying billions of different peptides areintroduced. Only those cells capable of expressing the proper smallpeptide that can block the VEGF-receptor interaction, and, consequently,the activation of the artificial negative gene, are allowed to grow andto form colonies on terrains containing the selective and specificchemical compound. DNA sequences encoding the active small peptide, theeffector peptide, are further characterised as described in the p53 caseabove.

Screening for agonists in view of a defective and thus not interactingprotein

As already mentioned above, also peptides “healing” the absence of adesired protein—protein interaction action due to a defect in one of theinteracting proteins can be screened. In this case, the host cell istransformed formed as described above for the search for antagonistswith the exception that the transformation is performed with a “correct”and a “defective” sequence encoding a correct and a defective protein,one of them being linked to a DNA-binding unit, the other one beinglinked to an activation unit. The monitoring system in this casecomprises at least one selector/reporter gene which enables the survivalof the cell, if expressed. Such a selector/reporter gene can be one asdescribed with regard to the agonist system.

Also, such cells are brought into contact with the DNA library, and onlythose cells are able to survive on a specific terrain, which comprise apeptide able to restore the activity of the defective protein so that anactivating interaction between the two proteins is possible, saidinteraction leading to the expression of a survival enabling monitoringprotein.

Also in such a system, the DNA of interest can be cloned and analysed asdescribed above.

Screening for antagonists against active protein

It is also possible that, due to a defect, a protein activating theexpression of an undesired gene is expressed in too large amounts, or inthe wrong cell, or at the wrong time.

In this case, a system similar to the first described system forscreening for an agonist can be applied, with the amendment, that aselector/reporter gene has to be used leading to cell death in theabsence of a protein-peptide interaction disrupting the proteinactivity.

Suitable activation systems:

Suitable activation systems for screening systems involving hybrids areknown (see e.g. above cited state of the art). Some of such activationsystems comprise a DNA sequence activating the transcription of aprotein upon contact with an activating protein (activation of pol II)or an activation complex (activation of pol III). Such activatingproteins comprise two essential parts, namely a DNA binding domain andan activation domain. Such polymerase II related activation systems aredescribed in the state of the art. In cases where such polymerase IIrelated activation systems are unsuitable, other systems can be used, inparticular systems related to polymerase III.

A polymerase III related system is described in Marsolier, Prioleau andSentenac, A RNA Polymerase III Based Two-hybrid System to Study RNAPolymerase II Transcriptional Regulators, J. Mol. Biol. 268, 243-249,1997. However, the system disclosed therein is not very reliable. Anamended system has therefore been developed, which is not only suitablefor the purposes of the here disclosed inventive screening system, butfor all two hybrids involving applications.

Polymerase III related activation system:

The pol III activation system is a complex comprising a DNA-bindingdomain and an activating protein, which interacts with a sub-unit of thetranscription apparatus (see Marsolier et al.).

Although the UASG-SNR6 promoter of the state of the art (see Marsolieret al.) offers the possibility to direct pol III transcription by aprotein—protein interaction, the difficulty of monitoring this eventremains. All experiments showing that this artificial transcriptionactivation works have been done by northern blot analysis. In theseexperiments, the UASG-SNR6 promoter controlled the expression of a“maxi” SNR6 gene in which 24 additional base pairs were inserted inorder to distinguish the wild type U6 RNA from the U6 maxi RNA in anorthern blot. Although northern blot analysis is sensitive to detectRNA transcripts, it is an unsuitable technique to perform a geneticscreen. An alternative system for selection of an activated UASG-SNR6promoter has thus been developed as follows.

A UASG-SNR6 promoter harbouring the wild type transcribed SNR6 sequence(reporter construct) was introduced into the yeast strain yMCM616, whosechromosomal SNR6 gene had been mutated. The strain yMCM616 survives witha wild type SNR6 gene, carried by a URA3-marked plasmid (survivalconstruct). Transformants containing either τ138-GAL DBD fusion alone orGAL4 and τ138 fused to interacting proteins became independent from thesurvival construct since they had the possibility to trigger SNR6transcription from the UASG-SNR6 reporter construct. The monitoring oftranformants containing interacting proteins was performed by selectingfor the loss of the URA3-marked plasmid. This selection can be done with5-fluoro-orotic acid (FOA), an uracil precursor analog, which ismetabolised to a toxic compound by the URA3 product. Only cells that areallowed to lose the URA3-plasmid (survival construct) can grow on FOA.Transformants without possibility to activate the UASG-SNR6 promoter,due to the lack of interaction between the proteins fused to GAL4 DBDand to τ138, were still dependent on the SNR6 gene carried by theURA3-marked survival construct and therefore incapable to grow on FOA.

It has now been observed that, while FOA is highly selective againstwild type yeast cells, overexpression of certain proteins leads to areduced selectivity and, consequently, to URA3 positive cells capable ofgrowing on FOA.

Thus, screening analysis of a genomic library with such a system wouldbe most likely susceptible to artifacts. So, there was a need togenerate a well controllable and highly selective screening system,based on the RNA polymerase III two-hybrid system.

Such an alternative to the FOA/URA 3 selection system, which is bettersuitable for a library screening, is obtained as follows. For thispurpose, yeast strains which are able to grow at 30° C. but not at 37°C. were generated. This temperature sensitivity is due to a mutation inan episomally expressed SNR6 gene which is under the control of itsnatural promoter (ts survival construct). The wild type SNR6 allele,whose expression can rescue the temperature sensitive phenotype, isintegrated into the genome, and is under the control of the artificialUASG-SNR6 promoter. As it is the case in the original pol III two-hybridsystem, the expression of this SNR6 reporter gene is dependent on theinteraction between the τ138 fusion protein (τ138-X) and the UASG-boundGAL4 fusion protein (Y-GAL4 DBD). In this novel and advantageousselection system, expression of the wild type SNR6 sequence allows cellgrowth at the non-permissive temperature of 37° C.

Monitoring systems:

Suitable selector/reporter systems leading to cell death or cellsurvival when activated are known (see e.g. URA 3, LYS 2, CYH 2, HIS 3,LEU 2, Kan.^(r)).

In order to enhance the security of the screening system, it ispreferred, that the monitoring system comprises two selector/reportersystems, one enabling the survival on a specific terrain, and forexample a second one enabling the survival on another terrain orexpressing a colouring protein allowing the visual selection of cells.

Since for the generation of a suitable host cell transformation withseveral DNA sequences is needed, it is preferred that allselector/reporter sequences are introduced into the same vector, so thatless transformation steps are needed to get a suitably transformed hostcell. This has the advantage that either a host cell with fewerselection markers can be used or that more selection markers areavailable for other purposes. A further advantage is that e.g. twosequences encoding different monitoring proteins can be connected to thesame transcription activation sequence.

The vectors used to transform the host cells are preferably such onesthat lead to an incorporation in the genome of the selector/reportersequences, whereby the target protein encoding sequences, as well as theDNA library, preferably are present in episomal form.

Effector molecules with desired biological activity can be screened inthat an inventive screening system is brought into contact with asuitable selection medium selectively allowing the survival of cellswith desired target protein-effector peptide interaction.

A screening system according to the present invention is produced inthat the host cells are transformed with

at least one monitoring gene enabling the detection of said host cellupon transcription of said at least one monitoring gene, said at leastone monitoring gene being directly or indirectly under the control of aspecific activation system and,

at least one DNA sequence coding for at least one target molecule, saidone or more target molecule(s) being selected from the group comprisingRNA sequences or proteins, said one or more target molecule(s) beingresponsible in their natural environment for the induction of theproduction and/or activity of an undesired protein or the omission ofthe production and/or activation of a desired protein, said at least oneDNA sequence coding for said at least one target molecule being underthe control of an host cell specific active promoter, preferably a hostcell specific promoter, and

a DNA library encoding peptides, wherein the DNA is under the control ofan active promoter.

In such a production process with adivated monitoring proteins more thanone nucleic acid sequence encoding a monitoring protein can beintroduced into the cell on the same vector and under the control of thesame specific activation system. Such proceeding is preferred in view ofstability of the system, the need of markers and the screeningselectivity.

A further important object of the present invention is a DNA libraryencoding peptides, wherein the peptide encoding nucleotide sequences areunder the control of a promoter active in a specific host cell, inparticular in a yeast cell. Such a DNA library is preferably produced inthat genomic DNA or cDNA is fragmented by digestion or, preferably,sonication and digestion, or by synthesis of random oligonucleotides,preferably single stranded oligonucleotides, in order to generatepeptide encoding sequences, said peptide encoding sequences then beingintroduced as such, or fused to a defined protein, into expressionvectors able to transform specific host cells, whereby the peptideencoding sequences are under the control of a promoter active in aspecific host cell, in particular in yeast cells.

Vectors comprising double-stranded random oligonucleotides can easily beproduced by generating a 3′ overhang and a blunt end. Such an overhangand blunt end are readily obtained by digestion with suitablerestriction enzymes.

The random single-stranded oligonucleotides carry at the 3′ end a knownsequence complementary to the 3′ overhang. This feature allows directligation of the single-stranded oligonucleotide with the 3′ overhangallowing the formation of the complementary strand by polymeraseactivity. The resulting double-stranded sequence contains a blunt endwhich is then ligated with the plasmid blunt end.

This method is not limited to the generation of a DNA library of thepresent invention, but can be applied to any cloning of randomsequences.

A further object of the invention are host cells, in particular yeastcells, comprising a polymerase III activated selection system with atleast one destroyed genomic sequence encoding an essential molecule,said host cells additionally comprising a temperature sensitive mutantof said destroyed sequence under the control of an active promoter, anda sequence insensitive to non-permissive conditions under the control ofa specific transcriptional activator selectively activated uponinteraction of two proteins. Such sequences insensitive tonon-permissive conditions are e.g. wild-type sequences.

EXAMPLES

General remarks:

All sequences mentioned herein are listed in 5′→3′-direction. They areadditionally listed in the SEQUENCE LISTING following the experimentalpart.

Example 1 Yeast expression vectors

DNA vectors for expression of peptides in yeast contain a selectableyeast nutritional marker, a replication origin for maintenance in yeastcells, a constitutive or inducible promoter for activation of genesencoding peptides, a transcription termination sequence, and bacterialβ-lactamase gene and origin of replication for plasmid growth in E.coli. Expression vectors disclosed herein bear the standard yeast 2 μmorigin of replication, the TRP1 selectable marker and one of thefollowing yeast promoters: the constitutively expressed ADH1 and ACTINpromoters, and the inducible GAL1 promoter (FIGS. 9, 10 and 11). Theseexpression vectors contain restriction sites immediately downstream ofthe translation initiation codon ATG for cloning DNA sequences encodingpeptides. Translation stop codons in all three reading frames arepresent downstream of these cloning sites.

pESBA-ADH (FIG. 9):

This expression vector was constructed as follows: oligonucleotidesAGCTTAACAAAATGGGCCCGCAGGCCTAACTAACTAAG (SEQ. ID. No. 13) andAATTCTTAGTTAGTTAGGCCTGCGGGCCCATTTTGTTA (SEQ. ID. No. 14) were kinasedusing T4 polynucleotide kinase (PNK) and annealed according to standardprocedures to form a double-strand (ds) oligonucleotide. This dsoligonucleotide contains the initiation codon ATG, ApaI/Bsp120I and StuIrestriction sites, stop codons TAA in all three reading frames, andcohesive ends compatible with ligation in open HindIII and EcoRI sites.The ADH1 promoter sequence was isolated from the plasmid PADNS (ref. C.Guthrie and G. R. Fink Ed., Guide to Yeast Genetics and molecularBiology, Academic Press, Inc. 1991) by BamHI and HindIII digestionfollowed by preparative electrophoresis in a 1% agarose gel. Thetranscription termination sequence of the ADH1 gene and part of theyeast TRP1 gene were isolated from the plasmid pGBT9 (obtained fromClontech, USA) by EcoRI and XbaI digestion followed by preparativeelectrophoresis in a 1% agarose gel. The expression vector backbone alsocarrying the remnant of the TRP1 gene was prepared by SphI and XbaIdigestion followed by Calf Intestinal Phosphatase (CIP) treatment andpreparative electrophoresis in a 1% agarose gel. These three DNAfragments plus the ds oligonucleotide were ligated together in oneligation step using T4 DNA ligase.

pESBA-ACT (FIG. 10):

Oligonucleotides CATGGGCCCGCAGGCCTAACTAACTAAG (SEQ. ID. No. 15) andTCGACTTAGTTAGTTAGGCCTGCGGGCC (SEQ. ID. No. 16) were kinased and annealedto form ds oligonucleotides containing the initiation codon ATG,ApaI/Bsp120I and StuI restriction sites, stop codons TAA in all threereading frames, and cohesive ends compatible with ligation in open NcoIand SalI sites. This ds oligonucleotide was cloned into the expressionvector pJP224 (J. Pearlberg, PhD thesis, Harvard University, Cambridge,Mass., 1995) which had been digested with NcoI and SalI restrictionenzymes and treated with CIP. This vector contains the ACTIN promoter, aunique NcoI site overlapping with the initiation codon ATG, a SalI siteand, immediately downstream of it, the transcription terminationsequence of the GAL11 gene.

pESBA-GAL1 (FIG. 11):

The expression vector pJG4-5 (R. Brent in Current Protocols in MolecularBiology, Chapter 20, 1997, John Wiley & Sons, Inc.) was digested withHindIII, treated with Klenow to fill in the DNA ends, and then digestedwith SacI to isolate its GAL1 promoter. pESBA-ACT was digested withNcoI, treated with Klenow, and then digested with SacI to cut out theACTIN promoter. Upon separation by electrophoresis and extraction fromthe agarose gel, the GAL1 promoter fragment and the pESBA-ACT backbonecured of the ACTIN promoter were ligated such as to regenerate the SacIsite and join the blunt HindIII and NcoI ends.

Example 2 Yeast reporter constructs

Plasmids carrying reporter genes are designed for integration into theyeast genome. Each plasmid bears two divergently transcribed genesseparated by a multiple cloning sequence (MCS). This cluster of uniquerestriction sites is used to insert the desired protein-bindingsequences. One of the two reporter genes is LacZ, which encodes theenzyme β-galactosidase commonly used to measure gene expression in yeast(C. Kaiser et al., Methods in Yeast Genetics, A. Cold Spring HarborLaboratory Course Manual, 1994 Ed.). The second gene encodes either anenzyme whose expression is required for growth of the host cell onappropriate, selective terrain, or another enzyme whose expression hasto be inhibited in order to allow growth of the host cell. Reporterplasmids constructed so far contain, in addition to the LacZ codingregion controlled by the GAL1 core promoter, either the selectable HIS3gene with its own core promoter, or the URA3 coding region controlled bythe SPO13 promoter (ref. M. Vidal, PNAS). Expression of the HIS3 gene isrequired for yeast growth on media lacking histidine. The level of HIS3gene expression can be modulated by varying the concentration in themedia of 3-aminotriazole (3-AT), a competitive inhibitor of the HIS3gene product. In contrast, expression of the URA3 gene can be toxic toyeast cells growing on media containing the drug 5-fluorotic acid(5-FOA). In this case, cells would grow on 5-FOA-containing media onlyupon inhibition of URA3 expression (see Guide to Yeast Genetics andMolecular Biology).

pESBA-HIS3:

The Lacz reporter plasmid pJP158 (J. Pearlberg, PhD thesis, HarvardUniversity) was digested with BglII and partially with BamHI to cut outthe URA3 sequence. The fragment of 6422 base pairs containing the LacZcoding region and the GAL1 core promoter was isolated by agarose gelelectrophoresis and recircularised by ligation of the compatible BglIIand BamHI sites. The HIS3 coding region together with a 3′ untranslatedsequence of about 630 base pairs were amplified by PCR using theupstream primer GGCAGTCGACATTATATAAAGTAATGTG (SEQ. ID. No. 17) and thedownstream primer GGCAGTCGACGGACACCAAATATGGCG (SEQ. ID. No. 18). ThepJP158-derived plasmid was digested with XhoI and SalI and treated withCIP. The larger fragment was isolated by agarose gel electrophoresis.The PCR product was digested with SalI and cloned into the SalI-XhoIdigested plasmid. Orientation of the insert was determined byrestriction enzyme analysis, and the correct clone with the HIS3 genetranscribed in the opposite direction of the LacZ gene was amplified inbacteria.

The oligonucleotides TCGACTCTAGAGCGGCCGCGAGCTCCCGCGGGCATGCAGATCTCCCG GGG(SEQ. ID. No. 19) and TCGACCCCGGGAGATCTGCATGCCCGCGGGAGCTCGCGGGCCGCTCTAGAG (SEQ. ID. No. 20) were kinased and annealed to form dsoligonucleotides bearing a MCS and cohesive ends compatible withligation into an open SalI site. These ds oligonucleotides were insertedat the unique SalI site of the plasmid described above. Sequenceanalysis was performed to determine the orientation of the inserted MCSand to confirm that only one SalI site was restored upon insertion ofthe ds oligonucleotide.

pESBA-URA3:

A sequence containing the URA3 coding region fused to the SPO13 promoterdescribed in the paper of Vidal et al., was isolated by PCR using theupstream primer AGTTCAGTCGACGTATCCGTTTAGCTAGTTAG (SEQ. ID. No. 21) andthe downstream primer AATACTGCAGCAGTTTTTTAGTTTTGCTGGCC (SEQ. ID. No.22). These primers were designed such as to introduce a SalI site and aPstI site at the 5′ and 3′ ends of the PCR product, respectively. TheHIS3 coding region and promoter present on the vector pESBA-HIS3 werecut out by SalI and PstI digestion and substituted with the SPO13-URA3PCR product digested with SalI and PstI.

pESBA-URA3-17mer:

Oligonucleotides CGGAAGACTCTCCTCCGT (SEQ. ID. No. 23) andCTAGACGGAGGAGAGTCTTCCGCATG (SEQ. ID. No. 24) were kinased and annealedto form ds oligonucleotides bearing two binding sites for thetranscriptional activator GAL4 (17mer) and cohesive ends for ligationinto open XbaI and SphI sites. pESBA-URA3 was digested with XbaI andSphI, treated with CIP, extracted with phenol, and precipitated withethanol. The ds oligonucleotide described above was ligated into thisplasmid backbone with T4 DNA ligase, and the correct sequence wasconfirmed by sequence analysis.

Example 3 Peptide libraries

Peptides are encoded by random DNA sequences of different length clonedinto the yeast expression vectors described above. These random peptidesare either expressed as such or fused to defined proteins.

Peptide expression from random synthetic oligonucleotides:

Oligonucleotides were synthesised such as to contain random nucleotidesequences of variable length (from about 15 to about 60 nucleotides)followed, at their 3′ ends, by the conserved sequence GGCC. Expressionvectors described above were digested with ApaI (GGGCC′C) and StuI(AGG′CCT), and subsequently purified by agarose gel electrophoresis.These linearised vectors were incubated with a 20 fold molar excess ofrandom oligonucleotides in a 30 μl reaction mixture containing dNTP's (5mM each), standard T4 ligase buffer, 50 mM NaCl, 1 μl T4 ligase gase and1 μl Klenow (purchased from NEB). After incubation at 16° C. for 4 hoursand at room temperature for 30 minutes, the reaction mixture wasextracted with phenol and ethanol-precipitated in the presence of 2Mammonium acetate to selectively discard excess free oligonucleotides.DNA was resuspended in 10 μl water and used to transform bacteria.

Peptide expression from random genomic sequences:

Genomic DNA from various organisms such as yeast, Drosophila and mousewas isolated by standard procedure. Purified genomic DNA was sonicatedsuch as to obtain fragments of 300-500 base pairs. After phenolextraction and ethanol precipitation, these fragments were digested witha cocktail of the restriction enzymes AciI, AluI, BfaI, NlaIII, HhaIHaeIII, RsaI, and Sau3A, and subsequently treated with T4 DNA polymerasein the presence of 100 μM of each dNTP to make all DNA ends blunt.Expression vectors described above were digested with Bsp120 I (G′GGCCC)(MBI Fermentas) and treated with Klenow to fill in the open Bsp120 Iends. The vector was then digested with StuI, extensively treated withCIP, and purified by agarose gel electrophoresis. The so preparedvectors and the blunt-end genomic DNA fragments were ligated by T4 DNAligase according to standard protocol.

Example 4 Agonistic action to the ESBATech screening system

The agonistic action of the inventive screening system has been shownfor the tumor suppressor gene p53. The tumor suppressor gene p53 ismutated in more than 50% of all human cancers which results in theinability to properly bind to the p53 binding site sin target genesresponsible to induce cell cycle arrest or apoptosis. The four mostcommonly found mutations occur in the DNA binding domain of p53 and leadto a single amino acid substitution. For one such mutation (p53 R273H),it has been shown that a synthetic 22 amino acid long peptide (22merpeptide 46), stemming from the carboxy-terminal part of p53 (361-382)when fused to the Drosophila Antennapedia internalisation domain (17amino acids), can restore DNA binding activity of the mutated p53(Selivanova et al., Restauration of the Growth Suppression Function . .. , Nature Medicine, Vol. 3 No. 6, 632 ff., 1997). The present inventorsnow found that the 22 amino acid long peptide, when expressed from thepESBA-ADH or pESBA-ACT vector, is sufficient to partially reactivatemutated p53 R273H in transactivation experiments. This opens thepossibility to use the peptide libraries according to the presentinvention to screen for peptides which restore the mutated p53 to wildtype activity or to use this system to screen for agonistic actions ofpeptides in general.

Cloning of wild type and mutated p53

Wild type p53 has been cloned by PCR into the HindIII restriction sitesof the yeast expression vector pGAD424 obtained by Clontec, using thefollowing primers; upstream GCACAAGCTTACCATGGAGGAGCCGCAGCTAG (SEQ. ID.No. 1); downstream GCGTCAAGCTTTCAGTCTGAGTCAGGCC (SEQ. ID. No. 2). Aminoacid changes were introduced in p53, covering the four most commonlyfound mutations in cancer cells (Hollstein et al., Science 253, 49,1991). These mutations had been introduced by PCR using degeneratedprimers:

R175H: upstream GGAGGTTGTGAGGCACTGCCCCCACC (SEQ. ID. No. 3); downstreamGGTGGGGGCAGTGCCTCACAACCTCC (SEQ. ID. No. 4) R248W, R249P: upstreamGGGCGGCATGAACTGGCCACCCATCCTCACCATCATCAC (SEQ. ID. No. 5); downstreamGTGATGATGGTGAGGATGGGTGGCCAGTTCATGCCGCCC (SEQ. ID. No. 6) R273H: upstreamGGAACAGCTTTGAGGTGCACGTTTGTGCCTGTCC (SEQ. ID. No. 7); downstreamGGACAGGCACAAACGTGCACCTCAAAGCTGTTCC (SEQ. ID. No. 8) R282W: upstreamCCTGGGAGAGACTGGCGCACAGAGG (SEQ. ID. No. 9); downstreamCCTCTGTGCGCCAGTCTCTCCCAGG (SEQ. ID. No. 10)

Cloning of p53 binding sites

The four putative p53 binding sites were cloned (Bian and Sun, Proc.Natl. Acad. Sci. USA 94, 14753, 1997; El-Deiry et al., Cell 75, 817,1993; Kern et al., Science 256, 827, 1992) into the XmaI-SphI site ofpESBA-HIS (FIG. 12) using double stranded oligonucleotides with thecorresponding restriction enzyme overhangs resulting in pDE4xp53BS. Thesequence of the oligonucleotides is as follows: upstreamCCGGGACTTGCCTGGAAACATGTCCAGTAGACATGTTCTTAGGGCTTGCTTGCATG( SEQ. ID. No.11); downstream CAAGCAAGCCCTAAGAACATGTCTACTGGACATGTTTCCAGGCAAGTC(SEQ.ID. No. 12)

The reporter vector pDE4xp53BS was linearised at the HIS3 3′UTR usingXhoI and introduced into the yeast strain JPY9 (J. Pearlberg, Ph.D.thesis, Harvard University; A. Barberis et al., Contact with a Componentof the . . . , Cell. Vol. 81, 359-368, 1995), resulting in YDE4xp53.

Expression and transactivation of wild type and mutated p53

Yeast strain YDE4xp53 was transformed with the conventional lithiumacetate method (Methods in Yeast Genetics, a Cold Spring HarborLaboratory Course Manual, Cold Spring Harbor Laboratory Press, 1994)with wild type and mutated p53 expression vectors. Transactivationpotential of the different p53 molecules was determined by measurementof the LacZ expression on X-gal plates and liquid beta-galactosidaseassays as described (Escher and Schaffner, Gene Activation at a Distanceand Telomeric Silencing . . . , Mol. Gen. Genet. (1997) 256; 456-461).The expression level of wild type and mutated p53 was imonitored byWestern blot using PAb421 monoclonal antibody (Hupp and Lane, Curr.Biol. 4, 865, 1994). Wild type and mutated p53 showed same intracellularprotein level.

Example 5 Testing the antagonistic action of peptides in yeast

To test the antagonistic action of short protein fragments or peptidesin yeast, it was checked whether high production of a peptide couldinhibit a defined molecular interaction. For this purpose, theinteraction between GAL4, a yeast DNA-binding transcriptional activator,and GAL11P, a component of the RNA polymerase II holoenzyme, which hasbeen shown to activate transcription (A. Barberis et al., Contact with aComponent of the Polymerase II Holoenzyme Suffices for Gene Activation,Cell. Vol. 81, 359-368, 1995; S. Farrell et al., Gene activation byrecruitment of the RNA polymerase II holoenzyme, Genes & Development 10,2359-2367, 1996) was exploited. This protein—protein interaction and itseffect on gene expression could be specifically inhibited by expressinga short protein fragment from a high-copy plasmid (2 μm) in yeast.

The yeast strain used in these experiments is a derivative of JPY9(MATα, ura3-52, his3Δ200, leu2Δ1, trp1Δ63, lys2Δ385, gal4Δ11) (A.Barberis et al., Cell, Vol. 81, 359-368) in which the reporter constructpESBA-17mer-URA3 was integrated at the his3 locus. Integration throughhomologous recombination was piloted by linearisation of the reporterplasmid at the XhoI site prior to yeast transformation. Expression ofthe URA3 gene was exploited to select for stable integrants onura-media. Since basal level expression of the SPO13-URA3 fusion gene istoo low to allow cell growth on ura-media, yeast were co-transformedwith a plasmid vector expressing GAL4 which, by binding the 17mersequence, activated URA3 expression. Upon selection for integration ofthe reporter construct, this plasmid was readily cured from yeast cellsby negative selection for URA3 expression on media containing 5-FOA(ref. Guide to Yeast Genetics and Molecular Biology)

Activation of the divergent LacZ and URA3 reporter genes in the yeaststrain described above was induced by co-expression of the DNA-bindingdomain of GAL4 (amino acid residues 1-100) and GAL11P. GAL4(1-100) wasexpressed from the plasmid pRJR217 (Y. Wu et al., Quantification ofPutative Activator- Target Affinities . . . , EMBO J. 15, 3951-3963,1996), while GALllP was expressed from pSO₂₈ (A. Barberis et. al., CellVol. 81, 359-368). As expected, GAL4(1-100) did not activatetranscription in the absence of GAL11P. To inhibit theGAL4(1-100)-GAL11P interaction and, as a consequence of this, activationof the reporter genes, a peptide comprising the GAL4 residues 58-97,which are known to contain the GAL11P-interacting sequence, wasoverexpressed. This peptide was overexpressed from the vectorPESBA-ADH1. Inhibition of reporter gene expression was monitored bymeasurement of β-galactosidase activity in a standard ONPG assay, and bycell growth on media containing 5-FOA (Methods in Yeast Genetics, A.Cold Spring Harbor Laboratory Course Manual, 1994 Edition).

While there are shown and described presently preferred embodiments ofthe invention, it is to be distinctly understood that the invention isnot limited thereto but may be otherwise variously embodied andpractised within the scope of the following claims.

24 1 32 DNA Artificial sequence Upstream Primer 1 gcacaagctt accatggaggagccgcagct ag 32 2 28 DNA Artificial sequence Downstream Primer 2gcgtcaagct ttcagtctga gtcaggcc 28 3 26 DNA Artificial sequence UpstreamPrimer 3 ggaggttgtg aggcactgcc cccacc 26 4 26 DNA Artificial sequenceDownstream Primer 4 ggtgggggca gtgcctcaca acctcc 26 5 39 DNA Artificialsequence Upstream Primer 5 gggcggcatg aactggccac ccatcctcac catcatcac 396 39 DNA Artificial sequence Downstream Primer 6 gtgatgatgg tgaggatgggtggccagttc atgccgccc 39 7 34 DNA Artificial sequence Upstream Primer 7ggaacagctt tgaggtgcac gtttgtgcct gtcc 34 8 34 DNA Artificial sequenceDownstream Primer 8 ggacaggcac aaacgtgcac ctcaaagctg ttcc 34 9 25 DNAArtificial sequence Upstream Primer 9 cctgggagag actggcgcac agagg 25 1025 DNA Artificial sequence Downstream Primer 10 cctctgtgcg ccagtctctcccagg 25 11 56 DNA Artificial sequence Oligonucleotide 11 ccgggacttgcctggaaaca tgtccagtag acatgttctt agggcttgct tgcatg 56 12 48 DNAArtificial sequence Oligonucleotide 12 caagcaagcc ctaagaacat gtctactggacatgtttcca ggcaagtc 48 13 38 DNA Artificial sequence Oligonucleotide 13agcttaacaa aatgggcccg caggcctaac taactaag 38 14 38 DNA Artificialsequence Oligonucleotide 14 aattcttagt tagttaggcc tgcgggccca ttttgtta 3815 28 DNA Artificial sequence Oligonucleotide 15 catgggcccg caggcctaactaactaag 28 16 28 DNA Artificial sequence Oligonucleotide 16 tcgacttagttagttaggcc tgcgggcc 28 17 28 DNA Artificial sequence Upstream Primer 17ggcagtcgac attatataaa gtaatgtg 28 18 27 DNA Artificial sequenceDownstream Primer 18 ggcagtcgac ggacaccaaa tatggcg 27 19 50 DNAArtificial sequence Oligonucleotide 19 tcgactctag agcggccgcg agctcccgcgggcatgcaga tctcccgggg 50 20 51 DNA Artificial sequence Oligonucleotide20 tcgaccccgg gagatctgca tgcccgcggg agctcgcggg ccgctctaga g 51 21 32 DNAArtificial sequence Upstream Primer 21 agttcagtcg acgtatccgt ttagctagttag 32 22 32 DNA Artificial sequence Downstream Primer 22 aatactgcagcagtttttta gttttgctgg cc 32 23 18 DNA Artificial sequenceOligonucleotide 23 cggaagactc tcctccgt 18 24 26 DNA Artificial sequenceOligonucleotide 24 ctagacggag gagagtcttc cgcatg 26

What is claimed is:
 1. A screening system for peptide agonistscomprising a eukaryotic host cell stably transformed with a selectionsystem and an effector peptide expressing system, said selection systemenabling the survival of the cells in the case of a desired interactionbetween at least one defective target molecule and an effector peptidewith agonist activity, said desired interaction restoring the normalfunction of said defective are molecule whereby said selection systemcomprises (a) at least one monitoring gene enabling the detection ofsaid host cell upon transcription of said monitoring gene, said at leastone monitoring gene being directly or indirectly under the control of aspecific activation system and, (b) at least one DNA sequence coding forat least one defective target molecule, said at least one defectivetarget molecule being selected from the group consisting of RNAsequences and proteins, said at least one defective target moleculebeing responsible in its natural environment for the induction of theproduction and/or the activity of an undesired protein or the omissionof the production and/or the activation of a desired protein, said atleast one DNA sequence coding for said at least one defective targetmolecule being under the control of an host cell specific activepromoter, preferably a host cell specific promoter, whereby saidspecific activation system is selectively modulated in the presence ofat least one specific interaction between said at least one defectivetarget molecule and an effector peptide with agonist activity, wherebysaid specific activation system upon modulation directly or indirectlymodulates the transcription of at least one monitoring gene enabling thesurvival of said host cell, and whereby said effector peptide expressingsystem comprises a peptide encoding nucleic acid sequence under thecontrol of a host cell active promoter.
 2. The screening systemaccording to claim 1, wherein at least one of said monitoring genes is anucleic acid sequence encoding at least one monitoring protein enablingthe detection of said host cell upon expression of said at least onemonitoring protein, said at least one nucleic acid encoding said atleast one monitoring protein being under the control of said specificactivation system, and whereby said specific activation system uponmodulation modulates the expression of at least one monitoring proteinenabling the survival of said host cell.
 3. The screening system ofclaim 1, wherein the eukaryotic host cell is a yeast cell.
 4. Thescreening system of claim 1, wherein said at least one DNA sequencecoding for said at least one defective target molecule encodes a proteinthat is partially or completely inactive in its natural environment. 5.The screening system of claim 1, wherein said at least one DNA sequencecoding for said at least one defective target molecule encodes at leasttwo target molecules that upon interaction induce an undesired responsein their natural environment.
 6. The screening system of claim 1,wherein said at least one DNA sequence coding for said at least onedefective target molecule encodes a protein inducing an undesiredresponse in its natural environment.
 7. The screening system of claim 1,wherein said at least one DNA sequence coding for said at least onedefective target molecule encodes at least two target molecules, wherebyat least one of said molecules has a defect making a desired interactionwith at least one further target molecule impossible.
 8. The screeningsystem of claim 1, wherein the selection system comprises a firstnucleic acid sequence encoding a first monitoring protein and a secondnucleic acid sequence encoding a second monitoring protein, wherein thefirst nucleic acid sequence is a selector/reporter gene, which uponactivation or inactivation in a selection medium, enables cell survival,and the second nucleic acid sequence is a reporter gene, which uponactivation, expresses the second monitoring protein, which is a coloringprotein, said first and second nucleic acid sequences being under thecontrol of the same specific activation system.
 9. The screening systemof claim 1, wherein said effector peptide expressing system encodesmodifications of a specific peptide.
 10. A process for the screening ofeffector molecules with desired biological activity, wherein a screeningsystem according to claim 1 is brought into contact with a selectionmedium selectively allowing the survival of cells with desired targetmolecule-effector peptide interaction.
 11. A process for the productionof a screening system according to claim 1, comprising transforming hostcells with (a) at least one monitoring gene enabling the detection ofsaid host cell upon transcription of said at least one monitoring gene,said at least one monitoring gene being directly or indirectly under thecontrol of a specific activation system and, (b) at least one DNAsequence coding for at least one defective target molecule, said atleast one defective target molecule being selected from the groupconsisting of RNA sequences and proteins, said at least one defectivetarget molecule being responsible in its natural environment for theinduction of the production and/or activity of an undesired protein orthe omission of the production and/or activation of a desired protein,said at least one DNA sequence coding for said at least one defectivetarget molecule being under the control of a host cell specific activepromoter, and (c) a DNA library encoding peptides, wherein the peptideencoding regions of the DNA library are under the control of an activepromoter.
 12. The process of claim 11, wherein more than one nucleicacid sequence encoding a monitoring system is introduced into the cellon the same vector and under the control of the same specific activationsystem.
 13. The screening system of claim 1, wherein said at least oneDNA sequence coding for said at least one defective target molecule isunder control of a host cell specific promoter.
 14. The screening systemof claim 1, wherein said effector peptide expressing system comprises apeptide encoding nucleic acid sequence which is under control of a hostcell specific promoter.